Affinity purification and sequence determination of equine relaxin

Area of Science:

• Endocrinology and Reproductive Biology
• Protein Chemistry and Biochemistry
• Hormone Purification and Characterization

Background:

• Relaxin is a polypeptide hormone crucial for pregnancy across species.
• Previous purification methods for equine relaxin were time-consuming.
• Understanding relaxin's structure is key to its biological function.

Purpose of the Study:

• To develop a more rapid and efficient method for equine relaxin purification.
• To characterize the isoforms of equine relaxin.
• To determine the primary sequence of equine relaxin.

Main Methods:

• Affinity chromatography using monoclonal antibodies against equine relaxin.
• High-Performance Liquid Chromatography (HPLC) for isoform separation.
• Edman degradation and mass spectrometry for sequence determination.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE for structural analysis.

Main Results:

• A novel purification strategy combining affinity chromatography and HPLC was established.
• Four major and several minor isoforms of equine relaxin were isolated and characterized.
• Equine relaxin isoforms exhibited biological activity in the mouse interpubic ligament bioassay.
• The primary sequence was determined, revealing equine relaxin as the smallest sequenced relaxin (5253 Da) with A-chain (20 residues) and B-chain (28 residues).
• Isoform heterogeneity was attributed to the C-terminus of the B-chain.
• Equine relaxin showed 67% sequence identity with porcine relaxin.

Conclusions:

• Affinity chromatography coupled with HPLC provides an efficient method for equine relaxin purification.
• Equine relaxin exists as multiple isoforms due to C-terminal B-chain heterogeneity.
• The determined sequence and small size of equine relaxin offer insights into relaxin evolution and function.